We have continued studies on the role that energy-dependent protein degradation plays in regulating gene expression, using E. coli as a model system. We have found a role for the ClpXP and ClpAP proteases in the degradation of a novel set of unstable, abnormal proteins. The laboratory of Bob Sauer at MIT found that a stable RNA of E. coli called 10Sa RNA acts as a mobile message to direct the synthesis of an 11 amino acid C-terminal extension on the end of proteins when translation is truncated; the C-terminal tail targets the proteins for rapid degradation. We found that the cytoplasmic protease primarily responsible for the degradation is ClpXP; ClpAP also participates. DsrA, a small stable RNA that can overcome silencing of multiple genes in E. coli by the nucleoid-associated protein HNS, has been found to be necessary for the low temperature expression of a specialized sigma factor, RpoS. DsrA modulates RpoS synthesis by positively affecting translation of this protein. DsrA itself is synthesized preferentially at low temperatures (less than 30-C), and this temperature-sensitive synthesis may be sufficient to explain the low temperature expression of RpoS. RpoS is also rapidly degraded during exponential growth by the ClpXP protease; this degradation is in turn regulated by the response regulator protein RssB. We have found that RssB works on only the specific substrate, RpoS, since mutations in RssB that stabilize RpoS do not perturb degradation of another ClpXP substrate, lambda O protein. This suggests that environmental and cell cycle regulation via changes in protein degradation may operate by modifying substrate availability rather than protease activity.